SU896069A1 - Aspergillis insuetus g-116 strain as dextranase producent - Google Patents

Description

one

The invention relates to the microbiological industry and casas from a microorganism strain producing the dextranase enzyme used in the food industry during the filtration and purification of sugar syrups, as well as in medicine for the prevention and treatment of dental caries.

Various microorganisms are known that can synthesize dextranase from strains of various fungi and bacteria.

Known, for example, the strain Peuici & aum-f umicueosiom GEM-P129-producing dextranase. An enzyme is obtained in a liquid culture medium, at a pH value of 6.5-7, controlled by the addition of a kerlot to enhance the fermentation process and increase the yield of dextran G1 1.

. Closest to the proposed. technically and positively, the strain of the fungus PeuiciEBixnn piscarim 25 BIM G-102 is obtained. The level of extracellular endodextranase activity of this strain on Chapek-Doks medium with 1% dextran and 0.5% corn extract reaches 290 units / l on the 5th day 30

cultivation. Osaharivakid .. (exodextranase) extracellular activity is 32 U / ml 2,

A disadvantage of the known strains is the low level of extracellular dextranase activity.

The purpose of the invention is to obtain a strain of the fungus with a high level of extracellular dextranase activity.

The proposed strain AspergiECus inetuetus G-116 is an active single-pore variant of the original museum strain AspergiECus insuetus 99 HFVNII. The strain obtained by multiple sieving of the original culture on the environment with a specific inducer (native bacterial dextran), followed by the selection of active variants. Dextranase activity of the proposed strain is 2 times higher than the activity of the original. The strain is stored in the Museum of Cultures of the Institute of Microbiology of the Academy of Sciences of the Byelorussian SSR at h-4- + 5c on potato-dextrose agar (code of strain G-11b) and in the Museum of Cultures of Industrial Microorganisms of the Institute of VNIIGenetika (collection number CMPM G-136).

The strain Aspergi6 € us insuetus G-I6 possesses extracellular echodextranase activity up to 80 units / ml, enzodextranase activity of the culture fluid reaches 700 units / l.

The strain of the fungus AspergiEPus insuetus G-116 is characterized by the following cultural, morphological and biochemical characteristics.

Morphological signs. On the environment of čapek-Doksa, the vegetative mycelium is septate, branched, hyphae thickness of 3-4 microns. Conidiophores are non-septic, non-branching, direct. The conidial head is formed by a vertex swelling of the conidiophore 7–9–7–7 µm, a single layer of sterigm 12.5 .1b, µm and conidia chains, conidia are smooth, spherical, with a diameter of 4.5–6, 0 microns.

Cudtural signs. On Chapek-Doksa medium with sucrose on the 4th day at 28 ° C, the colonies were round, convex, with even edges, 2 cm in diameter, with thick short pubescence. The center of the colony is slightly raised / back side of the cream colored colony.

On the апapek-Doksa medium with dextran, the 4-day colonies are round, convex, high in the center, 3 cm long, the edges are even, the aerial mycelium is white, fluffy. In the center of the colony, the zone of sporulation is brown, 01.3 cm. Around the colony there is a clearance zone 5-6 mm wide.

On potato dextrose agar, the colonies are very convex with smooth edges. On the 4th day it has a diameter of about 2 cm, thick, short, white aerial mycelium. In the center of the colony is mycelium gray-green. On the 6th day, the diameter of the colony reaches 4 cm. Mycelium acquires a gray-green color in the center - gray-brown, spores are visible. On the edge of the colony the edge of the white aerial mycelium.

Biochemical signs. Aspergi 6us insuetus G-116 especially actively ferments glucose, sucrose, xylose, serbitol and dulcite, less active galactose, lactose, mannitol. Mildly assimilates glycerol and arabinose. Milk peptonizes very slowly, gelatine liquefies the cream, it is intensively hydrolyzed, you can get a real treat, you can get rid of it. nitrate and protein, forms of nitrogen. Nitrates restores.

The iilTaMM of the fungus Aspergi66us insuetus G-A16 is grown in a deep way on a medium with native dextran.

Extracellular enzyme dextranase activity is determined by viscometricity by the dilution ability of the enzyme, using as a substrate a 0.8% aqueous solution of native bacterial dextran. To do this, to 7.5 ml of a 0.8% dextran solution in 0.1 M Na-acetate buffer (pH 5.0)

0.5 ml of culture liquid is added (after appropriate dilution). At certain time intervals, the expiration time of the reaction mixture was measured on a VPZH-2 viscometer with a capillary diameter of 0.62 mm, thermostated at. Activity calculated by the formula

1 RA - -5 dtQ where A is the activity unit / l;

 substrate concentration;

v — relative viscosity;

t is the reaction time, s. Extracellular exodextranase activity is expressed in glucoen units. The unit of activity corresponds to the number of liberated by the action of the enzyme on the substrate of the recovery of four groups per 1 minute equivalent to one micromol of glucose. For this, to a 0.75 ml of a 0.15% dextran solution. In .. 0.1 M Na-acetate buffer (pH 5.0) 0.25 mm of culture liquid is added (after appropriate dilution). The hydrolysis is carried out for 10 minutes at the formed reducing sugars using the Samogy-Nelson method. Activity calculated by the formula CR

 10-ISO where A is the activity unit / ml;

C is the concentration of reducing substances in the reaction mixture,% µg / ml;

R is the dilution of the culture fluid;

10 - time of hydrolysis, min; 180 - mole of glucose.

Example. The cultivation of the strain AspergiCEus insuetus G-E6 is carried out on a nutrient medium containing,%: native dextran 1.0; NaNO. 0.3, KNaRS 4 0.1; KSE 0.05, MgSO4 0.05; RESID corn extract 0.5. Cultivation is carried out in flasks per 100 ml with 2 ml of medium on a shaker (200 rpm) at 28–29 ° C for 5 days.

The seed is a 2-week culture grown on potato-dextrose agar. Flushing from the surface of the agar (test tube for 25 ml of distilled water) in the amount of 4% of the volume of the sample, are added to the flasks. The maximum level of activity of the culture fluid was observed on the 5th day of cultivation. By this time, enzodextranase activity reaches 700 units / l, and exodextranene - BO units / ml.

A distinctive feature of the proposed strain Aspergi66us insuetus G-llti is the ability to actively grow and synthesize dextranase on a synthetic medium without adding

corn extract or other growth factors. At the same time, there are no significant differences in the level of dextranena activity of the culture liquid obtained on the medium with corn extract and synthetic medium.

(Itamm AspergiCCus insuetus G-116, as compared to the known, has a higher level of dextranase activity of the culture liquid by 2.5 times.

invention formula

The strain AspergiBEus insuetusГ-116 (collection of cultures of industrial microorganisms of the Institute of Scientific Research Institute of Collector Number Collection CMPM F-136), producing dextranase.

Sources of information taken into account in the examination

1. For Japan, 52-31429, cl. C 12 D 13/10, published 1977.

2, Authors' Certificate of the USSR I 747889, CP. C 12 D 13/10.

Claims (1)

Claim The strain AspergiSEus insuetus'G-116 (a collection of cultures of industrial microorganisms of the Institute of VNIIgenetics collection number CMPM F-136), a producer of 5 dextranases.